Figure 5. An MK2 substrate-specific p38 inhibitor blocks antiparalell pp-p38-MK2 heterodimer formation.

(A) Inhibition of MK2 and ATF2 by PoA or a generic (IoC/SB202190) p38 inhibitor was tested using MK2 and ATF2 phospho-specific antibodies in Western-blots. p38 activation in HT-M6 cells was initiated by doxycycline (DOX) addition. MKK6EE-FLAG expression was monitored by using anti-FLAG antibody and concomitant p38 activation was confirmed by using a pp-p38 specific antibody. (Inhibitors were added in 1 μM concentration an hour ago before sample collection.)

(B) The impact of inhibitors (1μM) on p38 mediated substrate phosphorylation was tested in in vitro kinase assays. Phosphorylation of a reporter construct (2 μM) - containing a p38 binding docking motif (MEF2A) and a MAPK phosphorylation target site (Garai et al., 2012) - was compared to MK2 (2 μM) phosphorylation. Concentration of pp-p38 was 10 nM. Kinase reactions were initiated by the addition of ATP(γ)³²P, reactions were stopped by the addition of loading buffer at different time points, samples then were loaded onto SDS-PAGE gels, and analyzed by phosphorimaging. Error bars show SD of the phosphorylation rates calculated based on three independent experiments.

(C) SAXS analysis of p38-MK2 complexes bound to the PoA inhibitor. SAXS data were collected on p38-MK2 and pp-p38-MK2 bound to PoA, and these were compared to binary complexes without the compound. The schematic below shows that PoA binding only affects the quaternary structure of the pp-p38-MK2 heterodimer. Inhibitor binding promotes the formation of the “inactive-like“ parallel complex from an antiparallel phosphorylated heterodimer, while the parallel nonphosphorylated p38-MK2 complex is unaffected. See also Figure S6.